Reagents and Methods for Appending Functional Groups to Proteins

Abstract

Methods and reagents for site-selective functionalization of peptides and proteins. The methods most generally involve the reaction of a thioester with hydrazine. Reagents include bifunctional reagents of formula:H2N—NH—CH2-M-L-FGand salts thereof where M is a single bond or a chemical group carrying a non-bonding electron pair, such as —C(O)NR′—, where R′ is H, or an alkyl or aryl group; L is an optional linker group as described above; and FG is a functional group having reactivity that is orthongonal to that of the hydrazine group. FG can, among others, be an azide, alkenyl, alkynyl, nitrile (—CN) or triazole group and is preferably an azide group (—N3). Methods and reagents can, for example, be combined with intein-mediated protein splicing to link proteins or fragments thereof to various chemical species or to a surface. Surface immobilization of proteins via the methods herein results in immobilized proteins which substantially retain biological activity and is thus useful for the generation of peptide or protein microarrays. Kits for functionalization and / or immobilization of peptides and proteins are provided as well as microarrays of peptides, proteins or both.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application takes priority from U.S. provisional application Ser. No. 60 / 820,011, filed Jul. 21, 2006, which is incorporated by reference in its entirety herein.STATEMENT REGARDING GOVERNMENT SUPPORT[0002]This invention was made with United States government support awarded by the following agencies: National Institutes of Health (NIH) GM044783. The United States government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein or peptide at a specific site with a functional group of orthogonal reactivity which can in turn be used for protein modification or immobilization.[0004]Site-specific derivatization of proteins and peptides is useful in a variety of research and therapeutic applications. For example, attachment of reporter molecules (labels or ta...

AI Technical Summary

Benefits of technology

[0017]This invention provides methods and reagents for site-selective functionalization of peptides and proteins. The method is based on the reaction of thioesters with hydrazine. The methods and reagents herein can be employed to covalently attach or link a peptide or protein, including protein fragments, to another chemical entity, which may be a functional group, a reporter molecule, label or tag, a biological molecule (e.g., a peptide, protein, carbohydrate, nucleoside or nucleic acid, or lipid), a ligand (for example, a steroid among many others) that in turn binds to a receptor (which may be a cell surface receptor), a variety of small molecules, e.g., drugs, drug candidates, antibiotics and the like, or a surface which most generally can be biological surface (e.g., a cell surface) or a substrate surface. The functionalization can be conducted in a physiological acceptable environment in aqueous buffer at pH near 7.0 (neutral pH) and at physiological temperatures which minimizes or avoids inactivation of biological molecules (e.g., peptides or proteins).

[0035]In specific embodiments, the invention provides a general method for producing proteins labeled site-specifically with an azido group. These azido-proteins can be produced by semisynthesis using a variation of expressed protein ligation (FIG. 1) [11]. The method involves producing the protein of interest as a fusion protein with an intein and a tag for affinity chromatography. On-resin cleavage of the intein-derived thioester is induced with a bifunctional reagent bearing a nucleophile for thioesters and an azido group, such as the bifunctional reagent above. This method is used to produce an azido-protein that maintains biological activity, preferably full biological activity, and displays at its C terminus an azido group that is available for chemoselective modification. Thus, exploiting the intrinsic and orthogonal reactivity of the thioester produced during intein-mediated protein splicing enables the site-specific chemical modification of a protein.

Problems solved by technology

While DNA microarrays [55] have been produced in a large scale to study gene function, the fabrication of protein microarrays is more arduous.

A major complicating factor is the tremendous chemical complexity of proteins.

In addition, proteins have limited stability and are susceptible to loss of activity when subjected to chemical modification.

Accordingly, one of the major challenges in protein microarray technology is the development of general and facile strategies for protein immobilization.

Although thiols are potent nucleophiles for thioesters, the resultant thioesters are inherently unstable to hydrolysis [9], making the simple transthioesterification of an intein-derived thioester unsuitable for the chemical modification of proteins.

This method, although site-specific, is labor intensive and low yielding.

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